1. Field of the Invention
The present invention relates to the detection, identification and monitoring of submicron size particles. More particularly, the invention pertains to a device and method for automated detection, identification, and monitoring which samples submicron size particles. Preferably, the present invention provides for the sampling, detection and identification of viruses and virus-like agents (such as, for example, prions, viral subunits, viral cores of delipidated viruses, etc.) in bioaerosols and fluids, especially biological fluids.
2. Brief Description of the Related Art
Detection and identification of viruses without limiting the detection and identification to a particular family, genus and species is extremely difficult. Searching for viruses pathogenic to humans in a single environment is also extremely difficult Additionally, sampling viruses and virus-like agents in air, as well as other fluids, increases the complexity of the detection and monitoring.
The difficulty of detecting and monitoring a wide range of viruses also varies by environment, but perhaps the most troublesome environment involves combat conditions. In particular, the problem of detecting and monitoring viruses in a potential biological warfare (BW) threat environment is extremely demanding. Notwithstanding the variation in virulence from virus to virus, a generally accepted figure is that ingestion of 10.sup.4 virions constitutes a significant threat to a soldier who breathes on the order of 1,000 liters (1 m.sup.3) of air per hour. Instruments with sensitivities which enable detection of remote releases of biological agents in a field environment thereby providing early warning capabilities, allowing calculations for troop movements and wind patterns, have been previously lacking.
Additionally, it has been previously difficult to maintain a broad-spectrum system for the detection of viruses which is free from false negatives because of natural or artificial mutations. The high mutation rates of known viruses, as well as the emergence of new viruses, such as the Ebola virus, must be addressed by any detection method. The potential for deliberate artificial mutations of viruses also exists. Furthermore, there are virus-like infectious agents, such as prions, which are suspected in causing scrapie, "mad-cow disease" and Creutzfeldt-Jakob disease. These prions possess no DNA or RNA, and can withstand 8 MRads of ionizing radiation before losing infectiousness. Other virus-like infectious agents, such as satellites, possess no proteins. However, detection of all of these agents must be possible for a device or method to be generally effective in the detection and monitoring of viruses and similar agents (such as, for example, virus fragments, prion, satellites, etc.) which are pathogenic to humans.
Detection and monitoring of viruses must also be free from false positives associated with background. Background includes biological debris which obscures the detection of the viruses by registering as a virus with the detection methods used in analyzing the samples collected. Analysis of viruses requires a very high degree of purification of those viruses to overcome background loading in order to avoid false positives. For example, a BW virus may be buried within loadings of other microorganisms which form biological debris having loading on a magnitude of 10.sup.10 larger than the threshold loading for the targeted virus itself.
Although methods that culture viruses can often be used to increase the virus over background, culture methods are too slow for efficient viral BW detection; furthermore, some important viruses cannot be cultured by known methods, and in any case cell culture is a highly variable and inconsistent method.
Viruses may also be extracted from an environment and concentrated to an amount that is required for detection and monitoring, without requiring any culturing. Quite generally, in the detection of small amounts of viruses in environmental or biological liquids, it is necessary to both enrich the concentration of viruses many orders of magnitude (i.e., greatly reduce the volume of liquid solubilizing the viruses) and accomplish exquisite removal of non-viral impurities; in the presence of non-viral impurities, even the most sensitive detection methods generally require virus concentrations on the order of 10 femtomoles/microliter or more in the sampled liquid in order to reliably detect the viruses.
Sampling for airborne viruses is generally accomplished by collecting airborne particles into liquid, using a process such as air scrubbing, or eluting from filter paper collectors into liquid. Since collection and subsequent separation and detection methods are strongly affected by the adsorption of viruses to solids in aerosols and by solids-association in water, this poses stringent requirements on the design of the sampling of air for viruses.
In contrast, when sampling liquids for viruses, in many cases no special equipment or processes may be necessary in order to collect a sample; for example, in sampling blood for viruses, only a standard clinical hypodermic needle may be needed, and similarly for other body liquids. For sampling of bodies of water or other conveniently accessible liquids, sample collection may not be an issue at all, and in such cases the term "collector" is often applied to what is, in reality, a virus extraction step (such as collection on a filter).
Currently, there is no simple and rapid method or device for detection of viruses which are pathogenic to humans in a BW environment. Rapid detection translates into protection for soldiers, more reliable and simplified strategic planning, and validation of other BW countermeasures. Previously known detection methods using biochemical reagents are impractical in the field, even for trained virologists. Additionally, reagent-intensive approaches, such as multiplex PCR, low-strigency nucleic acid hyridization, and polyclonal antibodies, increase the incidence of false positives several hundred-fold, whether under highly idealized laboratory conditions or in the field. Additionally, the hypervariability, or rapid mutation, of viruses and emergence of new, uncatalogued viruses precludes methods based on biochemical assays, such as PCR, immunoassay, and the like, from achieving broad-spectrum detection--detection of all viruses regardless of identity, known or unknown, sequenced or unsequenced.
There is a need for a highly reliable automated system.
In view of the foregoing, improvements in the detection and monitoring of submicron particles have been desired. In addition to increasingly rapid time response for the detection and monitoring of a universal sampling of viruses, an automated system is needed. Computer control of instruments and data collection/interpretation provides advantages of increased operator safety, simultaneous multiple location detection, decreased operator training and a greater consistency of results.